Good evening evryone.
I am actually working on dsRNA mycoviruses; but very recently, de HiSeq on my sample revealed the presence of an ssRNA virus. Therefore, i would like anyone who can give me an acurate protocol to amply its ends.
Thank you in advance
Dear Daniel,
I had an experience with ssRNA and sequencing the 3'end (poly-A tail starting site). I used a kit called 3'RACE (link below), if you need to amplify the 5'end as well there is also 5'RACE kit available.
However, in order not to spend that much, you can simply design poly-dT oligo with an adaptor sequence at 5'-end for your cDNA preparation and then by using primers to your viral sequence and to the adaptor, you can amplify the region. (as shown in 3'RACE protocol)
If you don't know your ssRNA sequence (I doubt because you said that you tracked it with HiSeq), there are other approaches, such as cloning the unknown consturct into a plasmid and sequence it.
https://www.thermofisher.com/de/de/home/references/protocols/nucleic-acid-amplification-and-expression-profiling/cdna-protocol/3-race-system-for-rapid-amplification-of-cdna-ends.html
Best,
Filip
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